Finding rare genetic variations in large cohorts requires tedious preparation of large numbers of specimens for sequencing. We are developing a solution, called DNA Sudoku, to reduce prep time and increase the throughput of samples. By using a combinatorial pooling approach, we multiplex specimens and then barcode the pools, rather than individuals, for sequencing in a single lane on the Illumina platform. We have developed a protocol for quantifying, calibrating, and pooling DNA samples using a liquid-handling robot, which has required a significant amount of testing in order to reduce volume variation. I will discuss our protocol and the steps we have taken to reduce CV. For accurate decoding and to reduce the possibility of specimen dropout, it is important that the DNA samples are accurately quantified and calibrated so that equal amounts can be pooled and sequenced. We can determine the number of carriers in each pool from sequencing output and reconstruct the original identity of individual specimens based on the pooling design, allowing us to identify a small number of carriers in a large cohort.